Effect of albumin concentration and serum matrix on ischemia-modified albumin

Abstract

There is concern that ischemia-modified albumin (IMA) levels measured by albumin cobalt binding (ACB) assay reflect mainly albumin concentrations rather than myocardial ischemia.
Serum matrix and proteins were separated from a serum pool by a membrane filter. Two series of pools with albumin concentrations of 10, 20, 30, 40, 50, and 60 g/L were prepared either with human albumin or serum protein fraction. IMA values of these pools were measured in quintiplicate.
There was a strong negative correlation between IMA and albumin levels in both pools. IMA change corresponding to each 10 g/L difference in albumin concentration was 37% and 48% in these pools.
ACB assay reflects albumin concentrations rather than IMA. Primary predictor of IMA in serum matrix is albumin concentration.

Full text

Effect of albumin concentration and serum matrix on
ischemia-modified albumin
Aylin Haklıgör, Arzu Kösem, Mehmet Şeneş,Doğan Yücel ⁎
Department of Clinical Biochemistry, Ankara Training and Research Hospital, Ulucanlar Caddesi, Cebeci, Ankara 06340, Turkey
Received 22 July 2009; received in revised form 19 August 2009; accepted 10 September 2009
Available online 19 September 2009
Abstract
Objectives: There is concern that ischemia-modified albumin (IMA) levels measured by albumin cobalt binding (ACB) assay reflect mainly
albumin concentrations rather than myocardial ischemia.
Design and methods: Serum matrix and proteins were separated from a serum pool by a membrane filter. Two series of pools with albumin
concentrations of 10, 20, 30, 40, 50, and 60 g/L were prepared either with human albumin or serum protein fraction. IMA values of these pools
were measured in quintiplicate.
Results: There was a strong negative correlation between IMA and albumin levels in both pools. IMA change corresponding to each 10 g/L
difference in albumin concentration was 37% and 48% in these pools.
Conclusions: ACB assay reflects albumin concentrations rather than IMA. Primary predictor of IMA in serum matrix is albumin
concentration.
© 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Keywords: Acute coronary syndrome; Albumin; Albumin cobalt binding assay; Ischemia-modified albumin; Myocardial ischemia
Introduction
Ischemia-modified albumin (IMA) has been suggested for
early diagnosis of myocardial ischemia. During ischemia,
endothelial and extracellular hypoxia, acidosis, and free radical
injury induce structural changes in the N-terminal region of
human serum albumin. Thus ability of albumin to bind transition
metal ions such as cobalt, copper, and nickel is reduced [1].
The exogenous cobalt binding capacity of albumin is
measured by the albumin cobalt binding (ACB) assay. Gidenne
et al. [2] evaluated the analytical characteristics of the ACB
assay and they showed a negative relationship between the ACB
assay results and albumin concentration of the sample. They
reported that for each 10 g/L increase in albumin, IMA values
were decreased approximately 20 kU/L. However, according to
the reagent insert of a commercially available kit form of the
ACB assay (Inverness Medical Innovations), IMA values are not
affected by albumin in a concentration range of 30–55 g/L.
Different approaches have been proposed to eliminate the
effect of albumin concentration on IMA. Lee et al. [3] proposed
“albumin-adjusted IMA index”: IMA index = serum albumin
concentration (g/dL)× 23 + IMA (U/mL) −100. Lippi et al. [4]
suggested adjusting the results by use of median albumin values
of the population: (individual serum albumin concentration /
median albumin concentration of the population) × IMA. The
aim of the present study was to investigate the relationship
between albumin concentrations and IMA by changing only
native albumin concentrations in the same sample matrix and by
adding purified albumin to the deproteinized serum matrix.
Methods
We designed two parallel studies in which separated serum
protein fraction or commercially available purified human
albumin were added into the same protein-free serum matrix.
To our knowledge, there is no study investigating IMA and
A
vailable online at www.sciencedirect.com
Clinical Biochemistry 43 (2010) 345 –348
Abbreviations: ACB, albumin cobalt binding assay; DTT, dithiothreitol;
HSA, human serum albumin; IMA, ischemia-modified albumin.
⁎Corresponding author. S.B. Ankara Eğitim ve Araştırma Hastanesi, Klinik
Biyokimya Bölümü, Ulucanlar Cad., Cebeci, Ankara 06340, Turkey.
E-mail address: doyucel@yahoo.com (D. Yücel).
0009-9120/$ - see front matter © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.clinbiochem.2009.09.006

albumin relationship in this way in the literature. A serum pool
including 45 g/L albumin was prepared from the residual sera
obtained from routine chemistry studies. Serum proteins were
removed with an Amicon® Ultra-4 filter (cutoff: 10,000 Da;
Millipore Corp.) by centrifugation at 4000 × gat room tem-
perature for 45 min. The albumin concentration of the separated
serum matrix was undetectable with bromcresol green method
(Olympus Diagnostica GmbH). When the serum matrix was
analyzed by using a Helena® SAS-MX SP-10 protein electro-
phoresis kit (serum matrix was applied without dilution), the
albumin band was also undetectable on the agarose gel. We
prepared two series of pools, pools A and pools B, from the same
serum matrix by adding either human albumin (Sigma Aldrich)
or the separated protein fraction into the matrix, respectively.
Albumin concentrations of these pools were 10, 20, 30, 40, 50,
and 60 g/L. IMA was measured by manual ACB test [1] in
quintiplicate in these pools. Within-run imprecisions (n= 11) of
ACB assay at low (ΔA: 0.440) and high (ΔA: 0.637) IMA
containing serum pools were 5.92% and 8.17%, respectively.
DTT, NaCl and CoCl
2
·6H
2
O were obtained from Merck, and
human albumin was from Sigma Aldrich. Albumin concentra-
tions of the prepared pools were measured in duplicate with an
Olympus AU640 analyzer (Olympus Diagnostica GmbH). ACB
test was performed with a Shimadzu UV 120-01 spectropho-
tometer (Shimadzu Corporation).
The relationship between IMA and albumin levels was
analyzed by linear regression. Percent differences of the IMA
values corresponding to each 10 g/L change in albumin con-
centration were calculated. IMA results were adjusted by
multiplying with the ratio of (albumin concentration of analyzed
pool / baseline albumin concentration) and we also calculated
percent differences of the adjusted results for each 10 g/L change
in albumin concentration. All statistical analyses were made by
an “SPSS for Windows ver. 15.0”and an “Analyzed-it”
packaged statistics program.
Results
IMA results obtained from pools A and B are shown in
Table 1 and Fig. 1. There was a strong negative correlation
between IMA and albumin concentrations both in pools A and
pools B (r=−0.985; Pb0.001 and r=−0.984; Pb0.001,
respectively). Linear regression equations were y
A
(IMA) =
1.069 −0.0128 x
A
(albumin concentration), (95% confidence
interval [CI] for intercept: 0.977−1.161; 95% CI for slope:
[−0.01540−(0.0029)]) in pools A; and y
B
(IMA) = 1.317 −
0.0167 x
B
(albumin concentration), (95% CI for intercept:
1.117–1.353; 95% CI for slope: [−0.0216−(−0.0156]) in pools
B. Corresponding change of IMA values to each 10 g/L change
of albumin concentration was calculated by use of regression
equations, and percent differences of the IMA values from
baseline IMA values of serum and serum matrix were presented
in Table 1. Adjusted IMA values were not significantly changed
by albumin concentration in the range of 20–50 g/L (r=0.285;
P= 0.535), but the differences from the baseline were N10% in
all the pools.
Discussion
We observed that there is a strong, negative relationship
between albumin concentrations and IMA, and IMA values were
directly determined by albumin concentrations at all albumin
levels. Additionally, there is a complex relationship between
DTT and cobalt ions: DTT and cobalt give a strong reaction in
deproteinized serum matrix, and this reaction is linearly and
gradually decreased with increasing albumin concentrations.
Table 1
Effect of albumin concentration on IMA.
Albumin
concentration (g/L)
IMA (ΔA) Adjusted IMA Difference from
the original serum
Difference from
serum matrix
Serum matrix 1 0 1.053
Pools A
2 10 1.023 0.227 192.3 −2.9
3 20 0.791 0.352 126 −24.9
4 30 0.628 0.419 79.4 −40.4
5 40 0.532 0.473 52 −49.5
6 50 0.412 0.458 17.7 −60.9
7 60 0.348 0.464 −0.6 −67
Pools B
2 10 0.992 0.220 183.4 −5.8
3 20 0.889 0.400 156.9 −14.6
4 30 0.732 0.488 109.1 −30.5
5 40 0.477 0.424 36.3 −54.7
6 50 0.308 0.342 −12 −70.8
7 60 0.096 0.128 −72.6 −90.9
Pools A and B were prepared by adding human albumin or protein fraction in the same serum matrix, respectively. Adjusted IMA was calculated according to the
formula proposed by Lippi. Accepting the ΔA values of original serum and serum matrix as baseline, differences (%) corresponding to each 10 g/L change in albumin
concentration were determined. Mean (SD) baseline IMA for serum: 0.350 ± 0.015 ΔA; baseline IMA for serum matrix: 1.053 ± 0.016.
346 A. Haklıgör et al. / Clinical Biochemistry 43 (2010) 345–348

Another interesting point is absence of any reaction or reaction
color between DTT and cobalt ions when water is used instead of
serum or serum matrix in reaction conditions. However, when
human serum albumin (HSA) is added to this reaction mixture,
the reaction color is observable.
Decreased or increased serum albumin concentrations
resulting from hemodilution, hemoconcentration, and various
diseases or physiological conditions affect IMA levels [5,6].
Van der Zee et al. [7] performed IMA measurements in
symptom-limited exercise myocardial perfusion scintigraphy
and showed that albumin concentration was the only indepen-
dent predictor of IMA levels; there was a negative correlation
between IMA and albumin concentration, and hemoconcentra-
tion due to physical exercise resulted in an absolute decrease of
IMA. Transient coronary artery occlusion during percutaneous
coronary intervention has been considered to prove the merit of
IMA for the diagnosis of myocardial ischemia. However,
recently we demonstrated that IMA results reflect albumin
concentrations rather than myocardial ischemia also in percu-
taneous coronary intervention [8]. Results of the present study
are consistent with those reported previously. Zapico-Muñiz et
al. [9] reported that each 1 g/L increase in albumin causes
approximately 2.6% decrease in IMA values of within 35–45 g/L
the range of albumin. We checked this relationship in a
concentration range of 0–60 g/L of albumin and found that
each 10 g/L change of albumin results in different IMA values of
37% in A pools, and 48% in B pools from the baseline.
Bhagavan et al. [10] reported that sensitivity and specificity
of IMA for acute coronary syndrome were 88% and 94% at a
cut-point of 0.500 ΔA. Because of the negative relationship
between albumin concentration and IMA, in patients with
decreased albumin concentration type 1 error, and in patients
with relatively increased albumin concentration type 2 error,
can be seen. In the present study, in both series of pools, when
we considered the 0.500 ΔA as cut-point, IMA values of the
pools with albumin concentrationN40 g/L wereb0.500 ΔA, i.e.,
these pools would be assumed as “non-ischemic”. On the
contrary, IMA values wereN500 ΔA in the pools with albumin
concentrationsb40 g/L, and when the results were adjusted by
Lippi's formula, all the IMA values remained in non-ischemic
area (b0.500 ΔA). Because IMA value of original pool was also
“non-ischemic”(0.350 ± 0.015 ΔA; mean ±SD), Lippi's formu-
la could be considered as conceivable; however, the differences
from the baseline IMA values were N10% in all the pools.
On the other hand, commercially available kit forms and
manual method use different IMA units. The kit form gives
IMA values with arbitrary units (kU/L) instead of mass
concentration of IMA or absorbance unit. Currently. there is
not a universal calibrator for ACB assay. Transferability and
traceability of ACB assay are limited because of the absence of
calibrators based on the mass concentration of IMA. Standard-
ization of the ACB assay by a human albumin-based universal
calibrator may improve the assay performance.
In conclusion, albumin concentration is the primary
predictor of IMA in the serum matrix. IMA results directly
reflect albumin concentrations rather than modified albumin.
Therefore, currently we suggest measurement of IMA by
ACB assay as a useless effort for triage of acute coronary
syndromes.
References
[1] Bar-Or D, Curtis G, Rao N, Bampos N, Lau E. Characterization of the Co
2+
and Ni
2+
binding amino acid residues of the N-terminus of human albumin.
Eur J Biochem 2001;268:42–7.
[2] Gidenne S, Ceppa F, Fontan E, Perrier F, Burnat P. Analytical performance
of the albumin cobalt binding (ACB) test on the Cobas MIRA® Plus
analyzer. Clin Chem Lab Med 2004;42:455–61.
[3] Lee YW, Kim HJ, Cho YH, Shin HB, Choi TY, Lee YK. Application of
albumin-adjusted ischemia modified albumin index as an early screening
marker for acute coronary syndrome. Clin Chim Acta 2007;384:24–7.
Fig. 1. Effect of the albumin concentration on IMA. Plot of IMA values (ΔA,
y-axis) versus albumin concentration (x-axis). Pools A (top) and B (bottom) were
formed by adding either human albumin or the separated protein fraction into the
same matrix, respectively. (■) and vertical bars: mean ΔA and minimum −
maximum ΔA for pools A and B; (---): regression line.
347A. Haklıgör et al. / Clinical Biochemistry 43 (2010) 345–348

[4] Lippi G, Montagnana M, Salvagno GL, Guidi GC. Standardization of
ischemia modified albumin testing: adjustment for serum albumin. Clin
Chem Lab Med 2007;45:261–2.
[5] Gaze DC, Crompton L, Collinson P. Ischemia modified albumin
concentrations should be interpreted with caution in patients with low
serum albumin concentrations. Med Princ Pract 2006;15:322–4.
[6] Borderie D, Allanore Y, Meune C, Devaux JY, Ekindjian OG, Kahan A. High
ischemia-modified albumin concentration reflects oxidative stress but not
myocardial involvement in systemic sclerosis. Clin Chem 2004;50:2190–3.
[7] Van der Zee PM, Verberne HJ, Van Straalen JP, Sanders GTB, Van Eck-
Smit BLF, de Winter RJ, et al. Ischemia-modified albumin measurements
in symptom-limited exercise myocardial perfusion scintigraphy reflect
serum albumin concentrations but not myocardial ischemia. Clin Chem
2005;51:1744–6.
[8] Demir H, Topkaya BÇ, Erbay AR, Doğan M, Yücel D. Ischemia-
modified albumin elevation after percutaneous coronary intervention
reflects albumin concentration rather than ischemia. Ann Clin Biochem
2009;46:327–31.
[9] Zapico-Muñiz E, Santaló-Bel J, Mercé-Muntañola J, Montiel JA,
Martinez-Rubio A, Ordóñez-Llanos J. Ischemia-modified albumin during
skeletal muscle ischemia. Clin Chem 2004;50:1063–5.
[10] Bhagavan NV, Lai EM, Rios PA, Yang J, Ortega-Lopez AM, Shinoda H, et al.
Evaluation of human serum albumin cobalt bin ding assay for the assessment of
myocardial ischemia and myocardial infarction. Clin Chem 2003;49:581–5.
348 A. Haklıgör et al. / Clinical Biochemistry 43 (2010) 345–348